The sequence preference of gamma radiation mutagenesis using a novel in vitro model

Purpose: Identifying the sequence pattern in the radiation mutagenesis is essential for understanding the DNA molecular evolution. The present study aimed to establish an in vitro radiation mutagenesis model using naked DNA and to discover the sequence pattern of radiation-induced mutations. Materials and methods: Human immunoglobulin heavy-chain variable region gene HVB was chosen as the targeted sequence. DNA molecules dissolved in water were irradiated with gamma rays (25Gy, 1 Gy/min), and four polymerases with different fidelity were used respectively for amplification. Ultra-sensitive next generation sequencing platform “EasyMF” was then employed to detect the mutation rate of all sites. Statistical analysis was performed using SAS9.4 with alpha 0.05. Results: The main result of this study consisted of two parts. One was comparing of the effects of polymerase with different fidelity on ionizing radiation mutagenesis and establishing the in vitro model. Low fidelity DNA polymerases, Taq and Platinum® Taq DNA polymerase, could display the effects of radiation mutagenesis. However, due to the high error rate of Taq DNA polymerase, Platinum® Taq DNA polymerase was more suitable for studying the sequence pattern of radiation mutagenesis. Another was that mutation rates of individual nucleotides G, C and its doublet combination were significantly higher after radiation treatment. Conclusion: Our findings suggest the fidelity of polymerase could affect the radiation mutagenesis. The nucleotides of G, C and their doublet combination showed higher radiation mutability. The sequence analysis of the radiation-induced mutation was helpful for the study of the evolution of DNA molecules under radiation. In addition, the comparisons of the polymerases was helpful to understand the role of the polymerase in the DNA damage repair.